Edible bird&#39;s nest extract and method of extraction

ABSTRACT

The present invention relates to a method for preparing a bird&#39;s nest extract and the various extracts obtained from said method. In an aspect of the present invention, there is provided a method for preparing a bird&#39;s nest extract, the extract comprising at least one molecule obtained from edible bird&#39;s nest (EBN), the method comprising the steps of: (a) washing raw EBN; (b) filtering the washed EBN; (c) extracting the molecule from the EBN; and (d) isolating the molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/084,076, filed Sep. 11, 2018, which is a U.S. National Phaseapplication under 35 U.S.C. § 371 of International Patent ApplicationNo. PCT/SG2017/050117, filed on Mar. 10, 2017, which claims priority toSingapore Patent Application No. SG 10201601905R, filed on Mar. 11,2016, all of which applications are incorporated herein by reference intheir entireties.

TECHNICAL FIELD OF INVENTION

The present invention relates to a method for preparing a bird's nestextract and the various extracts obtained from said method.

BACKGROUND OF INVENTION

Edible bird's nest (EBN) is the nest made from the saliva of swiftletsnaturally found in the South-east Asian region. The abandoned nests areharvested from the wild or from specially built housing for swiftlets.It has been reported that EBN exhibited various bioactivities andnutritional value that include potential for mitogenic response,epidermal growth factor (EGF)-like activity, anti-influenza virus,haemagglutination-inhibitory activity, lectin-binding activity,improvement of bone strength and dermal thickness, and hormone content.Processing of EBN can be different depending on the application. Ongoinginvestigations have been carried out to elucidate the biological andmedical functions of the edible bird's nest.

Currently, EBN is used in the form of a soup or other drinks by boilingthe EBN in water and consuming. The molecules of EBN in such a scenarioare large biomacromolecules that are difficult for the body to digestand absorb. As a result, the bioavailability of the beneficialcomponents of EBN prepared in such a manner is low, and the beneficialeffects of EBN is not maximised.

However, consuming whole EBN may lead to immunoglobulin E (IgE) mediatedanaphylaxis (Goh et al., 2001, J. Allergy Clin. Immun., 107(6),1082-1088) and EBN is thought to be the most common cause offood-induced anaphylaxis which could be life-threatening among children.

Another problem with crude EBN is the presence of undesirable compoundseither due to natural causes or added intentionally during processing.Adulteration of EBN commonly takes place decreasing the quality of theEBN. Adulterants used include pig skin, agar, red seaweed and karayagum. In order to camouflage adulterants and waste matters, bleaches areoften added.

Of particular concern is the presence of nitrite salts which is derivedmainly from the faeces of the swiftlets. Nitrites can also be added towhite bird's nest during processing to turn it into red bird's nestwhich is commercially more valuable. Ingestion of excessive nitrites hadbeen linked to cancer (Bryan et al. Food Chem. Toxicol. 2012, 50 (10),3646-3654).

Viruses, bacteria and fungi could contaminate EBN in the wild or in thefactory during processing. Concerns with regards to avian flu in wildbirds can lead to restriction of imports of whole EBN itself.

Therefore, there is a need to improve the processing of EBN to improvethe overall quality and beneficial properties to the consumer. Byextracting and isolating desirable compounds from EBN, harmful effectsare avoided or minimised while maximising the therapeutic benefits ofEBN.

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

Any document referred to herein is hereby incorporated by reference inits entirety.

SUMMARY OF INVENTION

In a first aspect of the present invention, there is provided a methodfor preparing a bird's nest extract, the extract comprising at least onemolecule obtained from edible bird's nest (EBN), the method comprisingthe steps of: (a) washing raw EBN; (b) filtering the washed EBN; (c)extracting the molecule from the EBN; and (d) isolating the molecule.

Preferably, the washing step comprises exposing the EBN to a firstenzyme solution at ambient temperatures for about 5 minutes and soakingthe EBN and enzyme solution in water for a further 5 minutes. Morepreferably, the first enzyme solution comprises a nitrite reductase. Inan embodiment, the water may be obtained from a reverse osmosis process.The washing step may involve washing the EBN in oxygenated water forabout 10 minutes followed by a drying period for about 12 hours at 70°C.

Preferably, the method further comprising sterilising the washed EBNprior to the extraction step at 121° C. for about 10 minutes.Alternatively, the extraction process may be carried out for about 20minutes.

The extraction solution may comprising any one selected from the groupcomprising: an anti-N glycan, an anti-O glycan, an anti-sialic acid(particularly a sialic acid binding Ig-like Lectin 14), an anti-zincfinger, an anti-perlecan, an anti-helix-turn-helix, an anti-hyaluronan,an anti-decorin, an anti-dermatan, an anti-chondroitin, an anti-lumican,an anti-keratan, an anti-syndecan, an anti-leucine zipper, ananti-heparan sulphate, an anti-brevican, an anti-neurocan, ananti-versican.

By “anti-”, it is meant to refer to any molecule that selectivelytargets and binds to the target of interest, i.e. the target being aglycan (N or O glycan), a sialic acid (particularly an anti-sialic acidbinding Ig-like Lectin 14), an anti-zinc finger, an anti-perlecan, ananti-helix turn helix, an anti-hyaluronans, an anti-decorin, ananti-dermatan, an anti-chondroitin, an anti-lumican, an anti-keratan, ananti-syndecan, an anti-leucine zippers, an anti-heparan sulphate, ananti-brevican, an anti-neurocan, an anti-versican etc.

As such, it follows that the extraction solution of the presentinvention will give rise to an EBN extract comprising any one selectedfrom the group: an N glycan, an O glycan, an sialic acid (particularly asialic acid binding Ig-like Lectin 14), a zinc finger, a perlecan, ahelix-turn-helix, a hyaluronan, a decorin, a dermatan, a chondroitin, alumican, a keratan, a syndecan, a leucine zipper, a heparan sulphate, abrevican, a neurocan, a versican. These may be present in the extract inany suitable amounts. In various embodiments, the composition mayinclude any one of the following:

(a) EBN Extract 1

-   -   20% N glycans    -   30% O glycans    -   50% sialic acid binding Ig-like Lectin 14

(b) EBN Extract 2

-   -   70% zinc fingers    -   15% perlecans    -   15% N glycans

(c) EBN Extract 3

-   -   15% N glycans    -   25% helix turn helix    -   15% hyaluronans    -   35% decorin    -   10% dermatan

(d) EBN Extract 4

-   -   10% dermatan    -   15% chondroitin    -   55% hyaluronans    -   20% lumican

(e) EBN Extract 5

-   -   10% keratan    -   25% syndecans    -   35% chondroitin    -   30% hyaluronans

(f) EBN Extract 6

-   -   35% leucine zippers    -   30% decorin    -   35% lumican

(g) EBN Extract 7

-   -   25% heparan sulphate    -   15% brevican    -   20% neurocan    -   20% versican    -   20% decorin.

These amount percentages may be weight percentage of the entirecomposition or otherwise.

Preferably, in an embodiment, extracting the molecule is carried byexposing the washed EBN to any one of an extraction solution selectedfrom the group comprising:

-   -   (a) a solution comprising an anti-N glycan and an anti-O glycan;    -   (b) a solution comprising an anti-heparan sulphate, an        anti-chondroitin, an anti-keratan, and an anti-dermatan; and    -   (c) a solution comprising an anti-leuzine zipper, an        anti-helix-turn-helix, and an anti-zinc finger.

The amount present in each solution (a) to (c) above may be:

-   -   (a) 50% anti-N glycan and 50% anti-O glycan;    -   (b) 25% anti-heparan sulphate, 25% anti-chondroitin, 25%        anti-keratan, and 25% anti-dermatan; and    -   (c) 37% anti-leucine zipper, 33% anti-helix-turn-helix, and 30%        anti-zinc finger.

In addition, in alternative embodiments, the compositions of theextraction solution may be any one selected from the following:

(a) Composition 1

-   -   20% anti-N glycans (molecular weight between 20 kDa to 1000 kDa)    -   30% anti-O glycans (molecular weight between 30 kDa to 3000 kDa)    -   50% anti-sialic acid binding Ig-like Lectin 14 (molecular weight        between 20 kDa to 700 kDa); or

(b) Composition 2

-   -   70% anti-zinc fingers (molecular weight between 10 kDa to 200        kDa)    -   15% anti-perlecans (molecular weight between 50 kDa to 10000        kDa)    -   15% anti-N glycans (molecular weight between 20 kDa to 700 kDa);        or

(c) Composition 3

-   -   15% anti-N glycans (molecular weight between 20 kDa to 700 kDa)    -   25% anti-helix turn helix (molecular weight between 10 kDa to        300 kDa)    -   15% anti-hyaluronans (molecular weight between 100 kDa to 3000        kDa)    -   35% anti-decorin (molecular weight between 10 kDa to 200 kDa)    -   10% anti-dermatan (molecular weight between 50 kDa to 500 kDa);        or

(d) Composition 4

-   -   10% anti-dermatan (molecular weight between 50 kDa to 500 kDa)    -   15% anti-chondroitin (molecular weight between 30 kDa to 700        kDa)    -   55% anti-hyaluronans (molecular weight between 100 kDa to 3000        kDa)    -   20% anti-lumican (molecular weight between 20 kDa to 250 kDa);        or

(e) Composition 5

-   -   10% anti-keratan (molecular weight between 25 kDa to 500 kDa)    -   25% anti-syndecans (molecular weight between 20 kDa to 100 kDa)    -   35% anti-chondroitin (molecular weight between 30 kDa to 700        kDa)    -   30% anti-hyaluronans (molecular weight between 100 kDa to 3000        kDa); or

(f) Composition 6

-   -   35% anti-leucine zippers (molecular weight between 10 kDa to 300        kDa)    -   30% anti-decorin (molecular weight between 10 kDa to 200 kDa)    -   35% anti-lumican (molecular weight between 20 kDa to 250 kDa);        or

(g) Composition 7

-   -   25% anti-heparan sulphate (molecular weight between 30 kDa to        300 kDa)    -   15% anti-brevican (molecular weight between 10 kDa to 300 kDa)    -   20% anti-neurocan (molecular weight between 20 kDa to 300 kDa)    -   20% anti-versican (molecular weight between 50 kDa to 500 kDa)    -   20% anti-decorin (molecular weight between 10 kDa to 200 kDa).

Each composition may be different specificities, effects and hence,uses. A summary for each composition is as follows:

Composition 1

This unique composition provides an EBN's extract that at the minimalconcentrations of 0.5 g/l and 1 g/l could inhibit haemagglutination forH1N1 and H3N2 respectively. Similar extract with only sialic acid couldinhibit haemagglutination for H1N1 and H3N2 respectively ONLY at minimalconcentrations of 5 g/l to 160 g/l.

Secondly, for the in vitro infectivity of influenza H1N1, it is shownthat the addition of this composition at concentrations between 0.03 to2 g/l to mammalian cells reduced the virus titer generated by at least 2fold when compared to the culture without the extract compositionsupplementation. Similar extract with only sialic acid could only do soat concentrations 10 to 464 g/l, at much higher concentrations.

This shows that this extraction solution can extract a composition (i.e.the EBN extract) that is potent for the prevention of influenza viruses,much more potent than any sialic acid or compound in the market.

It is also shown that mammalian cells after 1 day infection with H1N1and H3N2 influenza viruses in the presence of minimal concentration of0.33 g/l, no cytopathic effects were observed.

Other competitors, i.e. other commercial EBN solutions were also testedin the haemagglutination inhibition assay with influenza A. These EBNsolutions were found to be either cytotoxic to the cells or did notexhibit any detectable anti influenza virus activity. In conclusion,GeneOasis' proprietary EBN extract is potent and superior in theprevention of influenza virus infection.

Composition 2

This unique composition provides an EBN extract that provides enhancedkidney cell growth in a concentration dependent manner when compared tothe control without this GO's proprietary EBN extract composition. Inthe presence of 3.3 g/l of this composition, kidney cells reached theconfluent cell density on day 3 (^(˜)5×105 cells/cm2) earlier than thecontrol (Day 4). The growth enhancement can be related to the presenceof active molecules found in this unique EBN extract.

Composition 3

This extraction composition provides an EBN extract that can elicit anincrease in human stem cell proliferation during cell expansion invitro.

Most importantly, comparing the present extract to Competitor A EBN byprotein amount revealed that 4.39% GeneOasis EBN can induce a cellgrowth curve that is statistically significantly different from 10%Competitor A EBN, starting from day 2 through day 5 of culture (***p<0.001 for all days; p values are shown on the graph). Similarly,comparing GeneOasis EBN (GOA) to Competitor A EBN by volume revealedthat 10% the present EBN extract can induce a cell growth curve that isstatistically significantly different from 10% Competitor A EBN,starting from day 2 through day 5 of culture (p values are shown on thegraph). This is significant and novel as it shows that the present EBNextracts are more potent than Competitor A EBN in (i) promoting cellgrowth and not causing cell death like that of Competitor A, and (ii)its ability to replace EGF.

Most importantly, comparing the present EBN extract (GOB) to CompetitorB EBN by protein amount revealed that 3.35% of the present EBN extractcan induce a cell growth curve that is statistically significantlydifferent from 10% Competitor B EBN, starting from day 4 through day 5of culture (** p<0.01 for day 4 and *** p<0.001 for day 5; p values areshown on the graph). Similarly, comparing the present EBN extract (GOB)to Competitor B EBN by volume revealed that 10% of the present EBNextract can induce a cell growth curve that is statisticallysignificantly different from 10% Competitor A EBN, starting from day 2through day 5 of culture (** p<0.01 for day 4 and *** p<0.001 for day 2and day 5; p values are shown on the graph). This is significant andnovel as it shows that the present EBN extracts are more potent thanCompetitor B EBN in (i) promoting cell growth, and (ii) its ability toreplace EGF, particularly at later stages of cell growth (day 4 and day5 of culture).

Composition 4

This composition provides an EBN extract that can elicit a statisticallysignificant increase in human stem cell proliferation during the courseof chondrogenic differentiation in vitro. This is novel as this is thefirst study to show not only an increase but also a statisticallysignificant increase in DNA (cellular proliferation).

Composition 5

This composition provides an EBN extract that elicits an increase inhuman stem cell chondrogenic differentiation in terms of proteoglycancontent in vitro.

Addition of the present EBN extracts lead to an increase inglycosaminoglycan (GAG) content per pellet and GAG/DNA ratio, mostlyfrom day 14 to day 28 of differentiation. The increase in GAG contentper pellet is most significant and most consistent from day 14 to day 28of differentiation using 2.5% to 5%. The increase in GAG/DNA ratio ismost significant and most consistent from day 14 to day 28 ofdifferentiation using 5% of EBN. GAG is a major type of proteoglycancommonly found in cartilage tissue.

Composition 6

This composition provides an EBN extract that leads to an increase inCollagen II content per pellet and Collagen II/DNA ratio, mostly fromday 21 to day 28 of differentiation. The increase in Collagen II contentper pellet is most significant and most consistent for 1.25% EBN fromday 7 to day 14, as well as for 2.5% EBN from day 21 to day 28 ofdifferentiation. The increase in Collagen II/DNA ratio is mostsignificant and most consistent from day 7 to day 28 of differentiationusing 1.25% EBN. Collagen II is a major type of collagen molecule orfibrils commonly found in articular cartilage tissue. Collagen IIcontent per pellet is indicative of the total functional output of stemcell-derived chondrocyte-like cells in vitro. Collagen II/DNA ratiosreflect Collagen II production per cell and are used as functionalindicators of how well the stem cells are differentiating intochondrocyte-like cells. Dotted lines refer to Collagen II per pellet andCollagen II/DNA ratios when no EBN extracts were added.

Composition 7

This extraction solution composition provides an EBN extract thatcontain EGF-like component, which can be beneficial to long termculturing of hNPC.

Preferably, the step of extraction is carried out in the presence of anacid.

Preferably, the extraction is carried out at between 25° C. to 37° C.for about 20 to 120 minutes.

Preferably, isolating the molecule is carried out in the presence of asecond enzyme solution. More preferably, second enzyme solutioncomprises a vegetable or fruit protease. The concentration of the secondenzyme solution may be between 10 ug/ml to 100 ng/ml. Further, the stepof isolating the molecule is carried out at 45° C. for 60 minutes at pH6.5 to 9.0.

Preferably, the isolation step further comprising heating the mixture at70° C. for 5 minutes to deactivate the enzymes in the second enzymesolution.

Preferably, the present method further comprising the step ofdehydrating the extracted mixture. More preferably, the dehydrating stepis freeze drying.

In a second aspect of the present invention, there is provided a bird'snest extract obtained from a method according to the first aspect of thepresent invention.

In a third aspect of the present invention, there is provided acomposition comprising an extract according to the second aspect of thepresent invention. Preferably, the composition further comprises amaltodextrin. In an embodiment, the amount of maltodextrin present inthe composition may be between 30 wt % to 75 wt %.

In an embodiment of the present invention, the extract may be used inmedicine. More particularly, the extract may be used in the manufactureof a medicament for the treatment and/or prevention of a conditionand/or disease.

Even more particularly, the extract may be used to improve the skin andtreat various skin ailments, dehydration and inflammatory skins, boostthe immune system, delay aging, promote metabolism, protect a person'svisual sight, improve blood circulation, regulate blood cholesterollevel, protect cardiovascular health, invigorate and renew cells, easearthritis discomfort, balance hormone levels, reduce incidence ofInflammation, control diabetes, treat degenerative joints, degenerativeskin, degenerative nervous system and the brain, and protect from kidneyfailure.

In a fourth aspect of the present invention, there is provided apharmaceutical composition comprising an extract according to the secondaspect of the invention a pharmaceutically-acceptable carrier, excipientor diluent.

Preferably, the composition or formulation is a unit dosage containing adaily dose or unit, daily sub-dose or an appropriate fraction thereof,of the active ingredient. The compositions of the present invention maynormally be administered orally or by any parenteral route, in the formof a pharmaceutical composition comprising the bird's nest extract,optionally in the form of a non-toxic organic, or inorganic, acid, orbase, addition salt, in a pharmaceutically acceptable dosage form.Depending upon the condition, disorder and patient to be treated, aswell as the route of administration, the compositions may beadministered at varying doses.

In human therapy, the bird's nest extract or compositions of theinvention can be administered alone but will generally be administeredin admixture with a suitable pharmaceutical excipient diluent or carrierselected with regard to the intended route of administration andstandard pharmaceutical practice. They may be administered orally (viatablets and capsules) or parenterally, for example, intravenously,intra-arterially, intraperitoneal, intrathecal, intraventricular,intrastemally, intracranially, intra-muscularly or subcutaneously, orthey may be administered by infusion techniques. They are best used inthe form of a sterile aqueous solution which may contain othersubstances, for example, enough salts or glucose to make the solutionisotonic with blood. The aqueous solutions should be suitably buffered(preferably to a pH of from 3 to 9), if necessary. The preparation ofsuitable parenteral formulations under sterile conditions is readilyaccomplished by standard pharmaceutical techniques well-known to thoseskilled in the art.

Compositions or formulations suitable for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

For oral and parenteral administration to human patients, the dailydosage level of the compounds of the invention will usually be from 1mg/kg to 30 mg/kg. Thus, for example, the tablets or capsules of thecompound of the invention may contain a dose of active compound foradministration singly or two or more at a time, as appropriate. Thephysician in any event will determine the actual dosage which will bemost suitable for any individual patient and it will vary with the age,weight and response of the particular patient. The above dosages areexemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited and such arewithin the scope of this invention.

Alternatively, the compositions of the invention can be administered inthe form of a suppository or pessary, or they may be applied topicallyin the form of a lotion, solution, cream, ointment or dusting powder.The compositions of the invention, particularly the bird's nestextracts, may also be transdermal administered, for example, by the useof a skin patch. They may also be administered by the ocular route,particularly for treating diseases of the eye. For application topicallyto the skin, the compounds of the invention can be formulated as asuitable ointment containing the active compound suspended or dissolvedin, for example, a mixture with one or more of the following: mineraloil, liquid petrolatum, white petrolatum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, they can be formulated as a suitable lotion or cream,suspended or dissolved in, for example, a mixture of one or more of thefollowing: mineral oil, sorbitan monostearate, a polyethylene glycol,liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

Generally, in humans, oral or topical administration of the compositionsof the invention is the preferred route, being the most convenient. Incircumstances where the recipient suffers from a swallowing disorder orfrom impairment of drug absorption after oral administration, the drugmay be administered parenterally, e.g. sublingually, buccally,transmucosal or transdermal means.

Currently, bioactive molecules like Chondroitin Sulfate (CS), KeratanSulfate (KS) and Hyaluronic Acid (HA) which are widely used insupplementing joint cartilage health are extracted mainly from animalparts or genetically modified microorganisms cloned in bioreactors.There is a lack of a standard for consistency in purity and yields andsafety aspects of the extracted products.

When these compounds are extracted from natural sources of edible birdnests (EBN) it resolves the issue of safety and sustainability, as EBNare very rich and abundant sources for such bioactive factors.

Chondroitin sulphate is used in arthritis treatment in the form ofcream, capsules and health supplement medications. Hyaluronic Acid isused widely in cosmetic and pharmaceutical industries globally.

Absorption through the skin and joint, capsules or through the oralroute of administration for such water soluble factors (CS), (KS) and(HA) has been a problem due to the poor permeability through hydrophobicmembranes or structures in the skin or intestine. Thus it is difficultfor the maximum and immediate effects of these compounds to reach therequisite sites in the body for millions of global sufferers of jointinflammation and pain. This has been a huge challenge for a sustainableand long term solution.

Through the process of separating compounds and their hydrolysisproducts such as chondroitin sulfate, hyaluronic acid, and keratansulfate, an economical method is provided to supply the compounds inhigh purity and variable dosage to suit the purpose. The productsisolated in this process is amenable to be delivered to the user in avariety of ways and methods to allow for effective and quick delivery ofthe compounds to the site of action. Especially with a suitableformulation, chondroitin sulfate, hyaluronic acid, and keratan sulfatecan be delivered in effective and useful doses to the sites of pain andinflammation.

Formulated in lipid forms, the present extract may be used to produce afirst economical product in the market that is transdermal and iscomposed of safe and sustainable bioactive molecules, extracted fromnatural but abundant supplies of EBN recycled crumbs.

In order that the present invention may be fully understood and readilyput into practical effect, there shall now be described by way ofnon-limitative examples only preferred embodiments of the presentinvention, the description being with reference to the accompanyingillustrative figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a flow chart of a method of preparing a bird's nest extractaccording to an embodiment of the present invention.

FIG. 2 Effect of SF-EBN on Vero cell growth. The presence of the SF-EBNin the culture medium showed significant improvement in cell growth(measured at day 3).

FIG. 3 SF-EBN from GeneOasis can prevent influenza virus (H1N1) mediatedhemagglutination. Based on the WHO recommended virus concentration of8HAU/50 ul, prevention of hemagglutination is obtained at SF-EBN of 0.5g/l (2⁶).

FIG. 4 Effects of SF-EBN from GeneOasis on the inhibitory effects ofinfluenza virus (H3N2) mediated haemagglutination. Based on the WHOrecommended virus concentration of 8HAU/50 ul, prevention ofhaemagglutination is obtained at SF-EBN concentration of 1.0 g/l (25).

FIG. 5 Haemagglutination assay for measuring the influenza virus titerin culture supernatant after infecting Vero cells in the presence ofSF-EBN. The assay was carried out 24 h post infection with H1N1 at MOIof 0.001. The dotted lines divide wells within a row wherehaemagglutination can be observed.

FIG. 6 Evaluation of SF-EBN from competitor B in in influenza virus(H1N1) mediated hemagglutination. H1N1 virus concentration of 16 HAU/50ul was used. SF-EBN of the of the present invention was diluted to matchthe soluble protein concentrations of SF-EBN from the competitor B.

FIG. 7 shows the effect of an EBN extract product obtained from theprocess described using Anti-N-glycans and/or Anti-Helix-Turn-Helix onhuman stem cell proliferation during cell expansion. The data in FIG. 7shows that the combination of 10% EBN extract and 3 growth factors havea similar positive effect on human stem cell proliferation as using 4growth factors. The 10% EBN can only replace the epidermal growth factor(EGF) and act as a substitute for EGF. EGF is not available as a foodsupplement and Pan et al. (Environ. Health Perspect., DOI:10.1289/ehp1409200) has shown that human EGF and parabens may lead toincrease proliferation of breast cancer cell lines. Therefore, an EBNextract would be a viable substitute for individuals who do not produceendogenous EGF needed for human mesenchymal stem cells (hMSC) activationand differentiation to chondrocytes.

FIGS. 8 and 9 shows a comparison between an EBN extract product obtainedfrom the process described using Anti-N-glycans and/orAnti-Helix-Turn-Helix on human stem cell proliferation compared to anEBN extract from competitor A and B. The effect of the EBN extract toreplace EGF is much more significant for the EBN extract compared toboth competitor A and B products.

FIGS. 10A-B Effect of EBN extract on proliferation and differentiationof hNPCs. Cell growth (A), cellular marker for proliferation (ki67) andneuronal differentiation (beta-Tubulin) (B) were measured after 1 weekof cultivation.

FIGS. 11A-B Long term effect of EBN extract on proliferation anddifferentiation of hNPCs. hNPCs were continuously cultured for threeweeks in presence of either growth factors and EBN extract. Afterward,hNPCs were harvested and seeded into new plates for cell growth study at20,000 cells/well. Cell growth (A), cellular marker for proliferation(ki67) and neuronal differentiation (beta-Tubulin) (B) were measuredafter 1 weeks of cultivation.

FIG. 12 Effect of EBN extract on morphology NPC cultures

FIG. 13 10% of the present EBN extract can replace EGF to achievesimilar growth curves of hMSC expansion in vitro. Statistical analysiswith two way ANOVA revealed that the cell growth of 3 Growth Factors+10%EBN is significantly different from that of 3 Growth Factors only,showing that 10% EBN elicits a positive effect by increasing cell growthfrom day 2 to day 4 of cell culture. In contrast, statistical analysiswith two way ANOVA revealed that the cell growth of 3 Growth Factors+10%EBN is not significantly different from that of 4 Growth Factor, showingthat 10% EBN can replace EGF to achieve similar hMSC growth from day 0through to day 4 of cell culture. p values, ** p<0.01, *** p<0.001 and**** p<0.0001.

FIG. 14 Addition of the present EBN extracts lead to an increase in DNAcontent per pellet, mostly from day 7 to day 14 of differentiation, withthe most significant and consistent results at day 7 with 2.5% to 5%EBN. Increase in DNA content is commonly used as a measure of cellularproliferation. Dotted lines refer to DNA content per pellet when no EBNextracts were added. Numbers refer to the fold change induced over 0% ofEBN. Boxes highlight those with fold increase more than 1.0.

FIG. 15 For the first independent experiment of n=3 pellets in total,addition of the present EBN extracts did not lead to a statisticallysignificant increase in DNA content per pellet from day 7 to day 28 ofdifferentiation. This is likely due to the variation in the DNA contentobserved between pellets of the same condition at each indicatedtimepoint. Increase in DNA content is commonly used as a measure ofcellular proliferation. Statistical analysis was performed with ordinaryone-way ANOVA with Tukey's multiple comparisons test. Blue boxeshighlight those that can be potentially significant if analysis can bedone on more samples. Red boxes highlight those that are statisticallysignificant.

FIG. 16 For the second independent experiment of n=4 pellets in total,2.5% and 10% of the present EBN extracts caused a statisticallysignificant increase in DNA content per pellet at day 7 ofdifferentiation, when compared to control conditions without any EBNextracts (0%). This shows that the present EBN extracts can elicit astatistically significant increase in human stem cell proliferationduring the course of chondrogenic differentiation in vitro. This isnovel as this is the first study to show not only an increase but also astatistically significant increase in DNA (cellular proliferation).Increase in DNA content is commonly used as a measure of cellularproliferation. Statistical analysis was performed with ordinary one-wayANOVA with Tukey's multiple comparisons test. Blue boxes highlight thosethat can be potentially significant if analysis can be done on moresamples. Red boxes highlight those that are statistically significant. pvalues, * p<0.05.

FIG. 17 For the combined results of n=7 pellets in total, 2.5% of thepresent EBN extracts caused a statistically significant increase in DNAcontent per pellet at day 7 of differentiation, when compared to controlconditions without any EBN extracts (0%). This shows that the presentEBN extracts can elicit a statistically significant increase in humanstem cell proliferation during the course of chondrogenicdifferentiation in vitro. This is novel as this is the first study toshow not only an increase but also a statistically significant increasein DNA (cellular proliferation). Increase in DNA content is commonlyused as a measure of cellular proliferation. Statistical analysis wasperformed with ordinary one-way ANOVA with Tukey's multiple comparisonstest. Blue boxes highlight those that can be potentially significant ifanalysis can be done on more samples. Red boxes highlight those that arestatistically significant. p values, * p<0.05.

FIG. 18 Addition of the present EBN extracts lead to an increase inglycosaminoglycan (GAG) content per pellet and GAG/DNA ratio, mostlyfrom day 14 to day 28 of differentiation. The increase in GAG contentper pellet is most significant and most consistent from day 14 to day 28of differentiation using 2.5% to 5%. The increase in GAG/DNA ratio ismost significant and most consistent from day 14 to day 28 ofdifferentiation using 5% of EBN. GAG is a major type of proteoglycancommonly found in cartilage tissue. GAG content per pellet is indicativeof the total functional output of stem cell-derived chondrocyte-likecells in vitro. GAG/DNA ratios reflect GAG production per cell and areused as functional indicators of how well the stem cells aredifferentiating into chondrocyte-like cells. Dotted lines refer to GAGper pellet and GAG/DNA ratios when no EBN extracts were added. Numbersrefer to the fold change induced over 0% of EBN. Boxes highlight thosewith fold increase more than 1.0.

FIG. 19 For the first independent experiment of n=3 pellets in total,addition of the present EBN extracts did not lead to a statisticallysignificant increase in GAG content per pellet and GAG/DNA ratio fromday 7 to day 28 of differentiation. This is likely due to the variationin the GAG and DNA content (refer to FIG. 15 ) observed between pelletsof the same condition at each indicated timepoint. GAG is a major typeof proteoglycan commonly found in cartilage tissue. GAG content perpellet is indicative of the total functional output of stem cell-derivedchondrocyte-like cells in vitro. GAG/DNA ratios reflect GAG productionper cell and are used as functional indicators of how well the stemcells are differentiating into chondrocyte-like cells. Statisticalanalysis was performed with ordinary one-way ANOVA with Tukey's multiplecomparisons test. Blue boxes highlight those that can be potentiallysignificant if analysis can be done on more samples. Red boxes highlightthose that are statistically significant.

FIG. 20 For the second independent experiment of n=4 pellets in total,addition of the present EBN extracts did not lead to a statisticallysignificant increase in GAG content per pellet and GAG/DNA ratio fromday 7 to day 28 of differentiation. This is likely due to the variationin the GAG and DNA content (refer to FIG. 15 ) observed between pelletsof the same condition at each indicated timepoint. However, 2.5% and 10%of the present EBN extracts could potentially lead to a statisticallysignificant increase in GAG content per pellet at day 7 ofdifferentiation, when compared to control conditions without any EBNextracts (0%). This could be due to the statistically significantincrease in DNA content per pellet (refer to FIG. 16 ) as GAG/DNAcontent remains unchanged. GAG is a major type of proteoglycan commonlyfound in cartilage tissue. GAG content per pellet is indicative of thetotal functional output of stem cell-derived chondrocyte-like cells invitro. GAG/DNA ratios reflect GAG production per cell and are used asfunctional indicators of how well the stem cells are differentiatinginto chondrocyte-like cells. Statistical analysis was performed withordinary one-way ANOVA with Tukey's multiple comparisons test. Blueboxes highlight those that can be potentially significant if analysiscan be done on more samples. Red boxes highlight those that arestatistically significant.

FIG. 21 For the combined results of n=7 pellets in total, addition ofthe present EBN extracts did not lead to a statistically significantincrease in GAG content per pellet and GAG/DNA ratio from day 7 to day28 of differentiation. This is likely due to the variation in the GAGand DNA content (refer to Figure. 2.2.2.) observed between pellets ofthe same condition at each indicated timepoint. GAG is a major type ofproteoglycan commonly found in cartilage tissue. GAG content per pelletis indicative of the total functional output of stem cell-derivedchondrocyte-like cells in vitro. GAG/DNA ratios reflect GAG productionper cell and are used as functional indicators of how well the stemcells are differentiating into chondrocyte-like cells. Statisticalanalysis was performed with ordinary one-way ANOVA with Tukey's multiplecomparisons test. Blue boxes highlight those that can be potentiallysignificant if analysis can be done on more samples. Red boxes highlightthose that are statistically significant.

FIG. 22 Addition of the present EBN extracts lead to an increase inCollagen II content per pellet and Collagen II/DNA ratio, mostly fromday 21 to day 28 of differentiation. The increase in Collagen II contentper pellet is most significant and most consistent for 1.25% EBN fromday 7 to day 14, as well as for 2.5% EBN from day 21 to day 28 ofdifferentiation. The increase in Collagen II/DNA ratio is mostsignificant and most consistent from day 7 to day 28 of differentiationusing 1.25% EBN. Collagen II is a major type of collagen molecule orfibrils commonly found in articular cartilage tissue. Collagen IIcontent per pellet is indicative of the total functional output of stemcell-derived chondrocyte like cells in vitro. Collagen II/DNA ratiosreflect Collagen II production per cell and are used as functionalindicators of how well the stem cells are differentiating intochondrocyte-like cells. Dotted lines refer to Collagen II per pellet andCollagen II/DNA ratios when no EBN extracts were added. Numbers refer tothe fold change induced over 0% of EBN. Boxes highlight those with foldincrease more than 1.0.

FIG. 23 Both 4.39% and 10% of the present EBN extracts are significantlybetter than 10% Competitor A EBN in inducing hMSC cell growth from day 2through day 5 of culture. Surprisingly, 3 Growth Factor+10% Competitor Ahas a cell growth curve that (i) is significantly different from that of3 Growth Factor only (p<0.05 for day 2, p<0.001 for all other days),(ii) is significantly different from that of 4 Growth Factor (p<0.001for all days), and (iii) is not significantly different from thatwithout growth factors (p>0.05 for all days). This shows that CompetitorA EBN (i) has an inhibitory effect that negates the effect of the other3 growth factors, (ii) cannot replace EGF to achieve a growth curvesimilar to 4 growth factors, and (iii) is not inducing cell growth whichalso implies that Competitor A EBN may be causing cell death instead.The statistical analysis for this set of results is not shown on thegraph and is accomplished separately. Most importantly, comparing thepresent EBN extracts (GOA) to Competitor A EBN by protein amountrevealed that 4.39% of the present EBN extracts can induce a cell growthcurve that is statistically significantly different from 10% CompetitorA EBN, starting from day 2 through day 5 of culture (*** p<0.001 for alldays; p values are shown on the graph). Similarly, comparing the presentEBN extracts (GOA) to Competitor A EBN by volume revealed that 10% thepresent EBN extracts can induce a cell growth curve that isstatistically significantly different from 10% Competitor A EBN,starting from day 2 through day 5 of culture (p values are shown on thegraph). This is significant and novel as it shows that the present EBNextracts are more potent than Competitor A EBN in (i) promoting cellgrowth and not causing cell death like that of Competitor A, and (ii)its ability to replace EGF.

FIG. 24 Both 3.35% and 10% of the present EBN extracts are significantlybetter than 10% Competitor B EBN in inducing hMSC cell growth, from day4 to day 5 and from day 2 through day 5 of culture respectively.Surprisingly, 3 Growth Factor+10% Competitor B has a cell growth curvethat (i) is significantly different from that of 3 Growth Factor onlyfrom day 2 through day 4 (p<0.01 for day 2, p<0.05 for day 3, p<0.001for day 4), (ii) is significantly different from that of 4 Growth Factor(p<0.01 for day 3, p<0.001 for day 4 and 5), and (iii) is significantlydifferent from that without growth factors (p<0.001 for all days). Thisshows that Competitor B EBN (i) has a significant positive effect oncell growth only from day 2 to day 4 when compared to that of 3 growthfactors only, (ii) cannot replace EGF to achieve a growth curve similarto 4 growth factors from day 3 through day 5 of culture, and (iii) isnot inducing cell death as compared to that of no growth factors. Thestatistical analysis for this set of results is not shown on the graphand is accomplished separately. Most importantly, comparing the presentEBN extracts (GOB) to Competitor B EBN by protein amount revealed that3.35% GeneOasis EBN can induce a cell growth curve that is statisticallysignificantly different from 10% Competitor B EBN, starting from day 4through day 5 of culture (** p<0.01 for day 4 and *** p<0.001 for day 5;p values are shown on the graph). Similarly, comparing the present EBNextracts (GOB) to Competitor B EBN by volume revealed that 10% of thepresent EBN extracts can induce a cell growth curve that isstatistically significantly different from 10% Competitor A EBN,starting from day 2 through day 5 of culture (** p<0.01 for day 4 and*** p<0.001 for day 2 and day 5; p values are shown on the graph). Thisis significant and novel as it shows that the present EBN extracts aremore potent than Competitor B EBN in (i) promoting cell growth, and (ii)its ability to replace EGF, particularly at later stages of cell growth(day 4 and day 5 of culture).

FIG. 25 depicts Oligomannose-type N-glycan, Hybrid-type N glycan,Biantennary complex-type N-glycan but not tri- and tetraantennarycomplex-type N-glycan, and Terminal α1-3-linked mannose.

FIG. 26 depicts Bisected di-, tridiantennary complex-type N-glycan, Tri-and tetraantennary complex-type N-glycan, and Tr- and tetraantennarycomplex-type N-glycan.

DETAILED DESCRIPTION OF INVENTION

In the present invention, a way to utilise EBN as a low cost source ofactive nutraceutical ingredients (ANIs) and yet having high efficaciessuch as active pharmaceutical ingredients (APIs) is presented.

Ambient or room temperature refers to a temperature in the range of 20to 30° C.

N-linked glycans refer to structures wherein the nitrogen in the sidechain of asparagine in the sequence Asn-X-Ser or Asn-X-Thr, where X isany amino except proline, forms a glycosidic bond with a glycan and theglycan may be composed of N-acetyl galactosamaine, galactose, neuraminicacid, N-acetylglucosamine, fucose, mannose, and other monosaccharides(Varki A, Cummings R D, Esko J D, et al., editors, Essentials ofGlycobiology, 2nd edition, Cold Spring Harbor (NY): Cold Spring HarborLaboratory Press; 2009).

O-linked glycans refer to structures wherein the oxygen in the sidechain of serine or threonine forms a glycosidic bond withN-acetyl-galactosamine which is attached to additional sugarmonosaccharides.

Heparan sulfate (HS) is a glycosaminoglycan defined by the disaccharideunit (GlcNAcα1-4GlcAβ1-4/IdoAα1-4)_(n), containing N- and O-sulfateesters at various positions, and typically found covalently linked to aproteoglycan core protein. The main disaccharide units that occur inheparan sulfate are shown structurally below.

Disaccharide Units Found in Heparan Sulfate

Abbreviations

GlcA=β-D-glucuronic acid

IdoA=α-L-iduronic acid

IdoA(2S)=2-O-sulfo-α-iduronic acid

GlcNAc=2-deoxy-2-acetamido-α-D-glucopyranosyl

GlcNS=2-deoxy-2-sulfamido-α-D-glucopyranosyl

GlcNS(6S) 2-deoxy-2-sulfamido-α-D-glucopyranosyl-6-O-sulfate

The most common disaccharide unit within heparan sulfate is composed ofD-glucuronic acid (GlcA) linked to N-acetylglucosamine (GlcNAc)typically making up around 50% of the total disaccharide units. Notshown are the rare disaccharides containing a 3-O-sulfated glucosamine(GlcNS(3S,6S) or a free amine group (GlcNH₃ ⁺).

Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) defined by thedisaccharide unit (GalNAcβ1-4GlcAβ1-3)_(n) with a general structuralformula I, modified with ester-linked sulfate at certain positions andtypically found covalently linked to a proteoglycan core protein. Achondroitin chain can have over 100 individual sugars, each of which canbe sulfated in variable positions and quantities.

Dermatan sulfate is a sulphated glycosaminoglycan like chondroitinsulfate wherein D-glucuronic acid is replaced by L-iduronic acid and thedisaccharide monomer is shown in general structural formula II.

Keratan sulfate (KS) is a linear polysaccharide that consists of arepeating disaccharide unit. Keratan sulfate occurs as a proteoglycan(PG) in which KS chains are attached to cell-surface or extracellularmatrix proteins. The basic repeating disaccharide unit within keratansulfate is (-3Galactoseβ1-4-N-acetylglucosamineβ1-). This can besulfated at carbon position 6 (C6) of either or both the Gal or GlcNAcmonosaccharides. However, the detailed primary structure of specific KStypes are best considered to be composed of three regions: a linkageregion, at one end of which the KS chain is linked to the protein, arepeat region, composed of the -3Galβ1-4GlcNAcβ1- repeating disaccharideunit and a chain capping region, occurring at the opposite end of the KSchain to the protein linkage region.

Hyaluronic acid is an anionic, non-sulfated glycosaminoglycandistributed widely throughout connective, epithelial, and neuraltissues. It is unique among glycosaminoglycans in that it isnon-sulfated, and can be very large with its molecular weight oftenranging in size from 5 to 20 kDa in vivo. Hyaluronic acid is a polymerof disaccharides, composed of D-glucuronic acid andD-N-acetylglucosamine, linked via alternating β-1,4 and β-1,3 glycosidicbonds.

Heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate,hyaluronic acid are types of N or O-glycans as the sugar parts join to aprotein at Asn or Ser/Thr.

Leucine zippers are a dimerization domain of the bZIP (Basic-regionleucine zipper) class of eukaryotic transcription factors. The bZIPdomains is 60 to 80 amino acids in length with a highly conserved DNAbinding basic region and a more diversified leucine zipper dimerizationregion. The leucine zipper is a common three-dimensional structuralmotif in proteins and has that name because every seven amino acids is aleucine in the dimerization domain.

In proteins, the helix-turn-helix (HTH) is a major structural motifcapable of binding DNA. It is composed of two α-helices joined by ashort strand of amino acids and is found in many proteins that regulategene expression.

A zinc finger is a small protein structural motif that is characterizedby the coordination of one or more zinc ions in order to stabilize thefold. In general, zinc fingers coordinate zinc ions with a combinationof cysteine and histidine residues. Originally, the number and order ofthese residues was used to classify different types of zinc fingers(e.g., Cys₂His₂, Cys₄, and Cys₆). More recently, a more systematicmethod has been used to classify zinc finger proteins instead. Thismethod classifies zinc finger proteins into “fold groups” based on theoverall shape of the protein backbone in the folded domain. The mostcommon “fold groups” of zinc fingers are the Cys₂His₂-like (the “classiczinc finger”), treble clef, and zinc ribbon (Krishna S S, Majumdar I,Grishin N V, 2003, Nucleic Acids Research 31 (2), 532-50).

For naturally occurring glycan-binding proteins (GBP), excluding glycanspecific antibodies, it is possible to classify GBPs broadly into twomajor groups—lectins and glycosaminoglycan binding proteins. Mostlectins are members of families with defined “carbohydrate-recognitiondomains” (CRDs) that apparently evolved from shared ancestral genes,often retaining specific features of primary amino acid sequence orthree-dimensional structure. Thus, new family members can be identifiedby searching protein sequence or structural databases. Despite thisability to predict new GBPs, the structures of glycans recognized bymembers of a single lectin family can be quite diverse.Single-site-binding affinities in many lectins appear to be low (withK_(d) values in the micromolar range), although some lectins recognizeglycans with much higher affinity (with K_(d) values in the nanomolarrange). For those lectins with low affinity, multivalent interactionsbetween multiple CRDs and multiple glycans are often required to producethe high-avidity binding interactions that are relevant in vivo. Lectinstend to recognize specific terminal aspects of glycan chains by fittingthem into shallow, but relatively well-defined, binding pockets. Incontrast, protein interactions with sulfated glycosaminoglycans seem toinvolve surface clusters of positively charged amino acids that line upagainst internal regions of extended anionic glycosaminoglycan chains.The lectins can be further categorised as R-type, L-type, P-type,C-type, I-type, and galectins. Examples of N-glycans recognised by somelectins are shown structurally. The determinants required for bindingare indicated in the boxed areas.

The general process to extract and isolate the ANIs from EBN is asfollows (shown in FIG. 1 ):

-   -   (a) Clean EBN to remove contaminants.    -   (b) The cleaned EBN was grounded and sifted through a mesh.    -   (c) The EBN powder was dissolved in water and treated with a        solution containing at least one antibody.    -   (d) The antibody and bound molecules may be separated    -   (e) The bounded molecules were hydrolysed partially with an        acidic solution, and the bounded molecules were released by        addition of peptides of large molecular weight.    -   (f) The released small molecules were isolated via dialysis.    -   (g) The isolated fraction was treated with an enzymatic solution        to further break down the compounds.    -   (h) After denaturation and removal of the enzymes, the isolated        fraction was dried to obtain a solid product.

The crude EBN (1 piece is approximately 10 to 50 g) is cleaned bysoaking in water to remove nitrites, mites and other contaminants. Theother possible contaminants that are removed may include heavy metals,bleach and other minute debris, including stains.

An effective method to remove the nitrites is to use a solutioncontaining nitrite reductase enzymes from fruits, plants and soil.Additionally, the solution may contain another enzyme to inactivate anyaccompanying bacteria that produce the nitrite. To remove mites, asolution containing special fruit proteases are used. Such examplesinclude any such protease from papaya (papain), kiwifruit (actinidin),pineapple (bromelain), fig (ficin) etc. These proteases may be used inany suitable concentration that will allow for the inactivation of thebacteria.

The EBN mixture was treated sequentially with each enzymatic solutionfor at least 5 minutes from room temperature to 40° C. Nanobubbling ofthe resultant suspension of EBN in the enzymatic solution will cause thedegraded cellular debris to float to the surface of the water where itcan be easily removed. The enzymatic solution is subsequently removedfrom the solid EBN. The solid EBN can be further washed to remove anyresidual enzymes and contaminants.

The cleaned EBN is dried to remove excess water, preferably at 70° C.for 12 h.

The cleaned EBN is grounded and sifted through a mesh. The size of themesh should be sufficient to remove any large impurities left,preferably in a size of 200 to 700 μm. Most preferably the mesh size is600 μm.

The EBN powder is placed in water, preferably distilled or deionisedwater, at 5° C. for 5 hours. A suitable concentration is 25 g of EBN in1000 mL of water. The mixture may be further sterilised at 121° C. for10 minutes if desired.

The EBN mixture is treated with an aqueous solution containing at leastone antibody in a temperature range from 4 to 37° C. for at least 20minutes. With a temperature of 25 to 37° C., 20 to 120 minutes suffice.With a temperature of 4° C., the mixture of antibody and EBN is kept forat least 9 hours. Generally, the lower the temperature the longer thetime required for the antibody solution to completely bind to thetargeted compounds.

The antibody/antibodies is selected from the following:

-   -   1. Anti-N-glycans    -   2. Anti-O-glycans    -   3. Anti-Heparan sulfate (sulphate)    -   4. Anti-Chondroitin sulfate (sulphate)    -   5. Anti-Keratan sulfate (sulphate)    -   6. Anti-Dermatan sulfate (sulphate)    -   7. Anti-Leucine zippers    -   8. Anti-Helix-Turn-Helix    -   9. Anti-Zinc fingers    -   10. A hyaluronic acid antibody

The antibody and bounded molecules can be separated from the mixture byany of the commonly known methods. Some of these methods includephysicochemical fractionation, class-specific affinity andantigen-specific affinity. Physicochemical fractionation includesdifferential precipitation, size-exclusion or solid-phase binding ofimmunoglobulins based on size, charge or other shared chemicalcharacteristics of antibodies. Class-specific affinity includessolid-phase binding of particular antibody classes (e.g. IgG) byimmobilised biological ligands that have specific affinity toimmunoglobulins. Antigen-specific affinity includes using specificantigens to purify antibodies through their specific antigen-bindingdomains.

The antibodies to be used may include naturally occurring antibodies, ormodified antibodies for example tagged antibodies that can facilitatethe separation of the antibodies. Some common examples of tags used withantibodies are His-tag and FLAG-tag. Additionally, the antibody used toextract the target molecule could be bound to a solid support. The solidsupport could be made of a ferromagnetic material or conventional inertsupport material. The antibodies are commercially available and can beused as such. If modifications of the antibodies are desired, there aremany methods as commonly known in the literature to modify theantibodies to obtain the desired characteristics. Antibody separation iscommonly employed to extract out target proteins in a “fishing” method.

After addition of antibodies for a period of time as describedpreviously, the mixture is treated with an acidic solution and heated to100° C. to cause partial hydrolysis of the target compounds. The acid ispreferably a food acid, for example citric acid, malic acid, aceticacid, tartaric acid, fumaric acid, and lactic acid. The mixture iscooled to room temperature and neutralised to a pH of 7.

The antibody bounded compounds are released from the antibody by addingexcess larger peptides of natural glycoaminoglycans and cellulartranscriptional regulators.

The released compounds are subsequently isolated from the addedpeptides, enzymes and antibodies via the use of a dialysis bag.

In another embodiment, the antibody solution may contain at least twoantibodies. For instance, a solution containing Anti-N-glycans andAnti-O-glycans, a solution containing Anti-heparan sulfate,Anti-chondroitin, Anti-keratan, and Anti-dermatan, a solution containingAnti-leucine zippers, Anti-Turn-Helix-Turn, and Anti-zinc fingers.

Preferably, the antibody solution composition are as follows whereby thepercentage given is the percentage weight of the antibody relative tothe total weight of antibody present in the solution:

-   -   1. 50% of Anti-N-glycans and 50% of Anti-O-glycans.    -   2. 25% of Anti-heparan sulfate, 25% of Anti-chondroitin, 25% of        Anti-keratan, and 25% of Anti-dermatan.    -   3. 37% of Anti-leucine zippers, 33% of Anti-Turn-Helix-Turn, and        30% of Anti-zinc fingers.

Alternatively, the mixture containing EBN can be sequentially treatedwith solutions containing a different antibody or antibodies, andseparated to extract out the desired compounds sequentially.

The isolated compounds can be further hydrolysed with vegetable and foodproteases at 45° C. for 1 hour at a pH of 6.5 to 9.0. The concentrationof enzymes used should be at least 10 μg/mL for effective hydrolysis.Preferably, the concentration of enzyme is up to 100 μg/mL, and corn ormaize terminal proteases are used. The enzymes are denatured by heatingthe mixture at 70° C. for 5 minutes. The enzymes precipitate out at atemperature above 55° C., hence the mixture can be filtered at atemperature above 55° C. to afford the desired compounds as a solutionin the filtrate.

The solution of desired compounds is dried to give the compounds as apowder. Preferably, the compounds are dried by freeze drying or spraydrying. The freeze drying is carried out by cooling the solution to atemperature between −180 to −70° C. with liquid nitrogen or dry ice, andsubmitting the frozen mixture to vacuum to sublime the ice. The freezedrying can be repeated if required to give a dried powdered product.

The dried powdered product can be mixed with other additives to give afood or pharmaceutical product. Alternatively, the product can bedissolved in water along with other additives.

Through the process of separating products such as chondroitin sulfate,hyaluronic acid, and keratan sulfate and their hydrolysis, an economicalmethod is provided to supply the compounds in high purity and variabledosage to suit the purpose. The products isolated in this process isamenable to be delivered to the user in a variety of ways and methods toallow for effective and quick delivery of the compounds to the site ofaction. Especially with a suitable formulation, chondroitin sulfate,hyaluronic acid, and keratan sulfate can be delivered in effective anduseful doses to the sites of pain and inflammation.

Examples of some possible formulations are provided where the percentagegiven is the weight percentage of the component relative to the totalweight of the product. The EBN extract in 25 to 70% by weight is mixedwith a sugar making up the remainder to 100%. A suitable sugar isdextrin or maltodextrin.

Some possible formulations are provided as follows:

-   -   1. 70% of EBN extract product and 30% maltodextrin.    -   2. 60% of EBN extract product and 40% maltodextrin.    -   3. 50% of EBN extract product and 50% maltodextrin.    -   4. 25% of EBN extract product and 75% maltodextrin.

The extracted product from EBN possess therapeutic or prophylacticbenefits and may be used in medicine or as preventive measures. Theextract may be used to improve the skin and treat various skin ailments,dehydration and inflammatory skins, boost the immune system, delayaging, promote metabolism, protect a person's visual sight, improveblood circulation, regulate blood cholesterol level, protectcardiovascular health, invigorate and renew cells, ease arthritisdiscomfort, balance hormone levels, reduce incidence of Inflammation,control diabetes, treat degenerative joints, degenerative skin,degenerative nervous system and the brain, and protect from kidneyfailure.

In Vitro Cellular Assay Results

In vitro cellular assay experiments were done to study the potentialtherapeutic effects of the isolated products from EBN and the resultsare discussed further here.

FIG. 2 shows the effect of 4 concentrations of an EBN extract productobtained from the process described using Anti-Zinc fingers on thegrowth of Vero cells. On Day 3, the EBN extract demonstrates that thevero cell growth is enhanced in a dose-dependent manner. Additionally,at the highest concentration (3.3 g/l) tested, the cell growth was atthe maximum and significantly higher than the negative control withoutthe addition of the EBN extract product. This indicates that the EBNproduct promotes Vero cell growth.

To determine the inhibitory effect of an EBN extract product obtainedfrom the process described using Anti-O-glycans on the ability ofhaemagglutin molecules located on the surface of the influenza virus tointeract with erythrocytes, a haemagglutination inhibition (HAI) assaywas performed. FIG. 3 shows the results of the HAI assay for the H1N1strain of influenza virus. Erythrocytes that do not bind with theinfluenza virus would settle to the bottom of the well and be observedas a red button. Erythrocytes that bind to the influenza virus wouldform a lattice. Haemagglutination was not observed until 2⁷ folddilution for the two virus concentrations tested (columns 1 to 6 a dotcan be seen in the well of the plate, all the columns left to the dottedline). The corresponding concentration of EBN extract at this dilutionis 0.5 g/l. In other words, 0.5 g/l concentration of this EBN extractproduct is able to inhibit the binding of the H1N1 influenza virus toerythrocytes.

FIG. 4 shows the HAI assay with the H3N2 virus strain. The concentrationof EBN extract product is 2.1 g/l and 1.0 g/l at virus concentrations of16 and 8 HAU/50 μL respectively.

FIG. 5 shows the effect of an EBN extract product obtained from theprocess described using Anti-O-glycans on the inhibition of influenzavirus infection of Vero cells. FIG. 5 shows the virus titerquantifications using HAI. Table 1 below shows that the EBN extractproduct reduce the infectivity of the H1N1 influenza virus on Verocells.

TABLE 1 Effect of EBN extract on H1N1 virus replication in Vero cells.Result of hemagglutination assays are derived from FIG. 5. Concentrationof Virus Titre Fold reduction EBN extract (g/l) (HAU/50 μL) ofinfectivity 0    10.7 ± 4.6  — 0.03 4 ± 0 2.6 0.26 5.3 ± 2.3 2   2   5.3 ± 2.3 2  

The effect of the EBN extract product obtained from the processdescribed using Anti-O-glycans on the inhibition of H1N1 viral activitywere compared to two commercially available EBN solutions (Competitor Aand B). The soluble protein content in the EBN extract solution,competitor A and B was determined by DC™ protein assay (Bio-Rad, Cat.No. 5000116) with bovine serum albumin as the protein standard. Thesoluble protein content of the three solutions were determined to be2021 μg/mL, 823 μg/mL and 628 μg/mL respectively (Table 3). Hence, theEBN extract solution obtained from the process was diluted to match therespective competitor solution. However, competitor A EBN solution wasfound to be cytotoxic to the cells and was not tested further, whilecompetitor B EBN solution showed no inhibition of the H1N1 virus. ThusFIG. 6 shows the comparison of the EBN extract solution of the presentinvention against competitor B's EBN solution.

TABLE 3 Physical properties of EBN solutions from competitor A and B.Soluble Protein Osmolarity Turbidity Content (μg/ml) (mmol/kg) (OD₆₀₀)pH GeneOasis 2021 348 0.067 8.6 Competitor A  823 187 0.056 6.7Competitor B  628 220 0.056 7.3

FIG. 7 shows the effect of an EBN extract product obtained from theprocess described using Anti-N-glycans and/or Anti-Helix-Turn-Helix onhuman stem cell proliferation during cell expansion. The data in FIG. 7shows that the combination of 10% EBN extract and 3 growth factors havea similar positive effect on human stem cell proliferation as using 4growth factors. The 10% EBN can only replace the epidermal growth factor(EGF) and act as a substitute for EGF. EGF is not available as a foodsupplement and Pan et al. (Environ. Health Perspect., DOI:10.1289/ehp1409200) has shown that human EGF and parabens may lead toincrease proliferation of breast cancer cell lines. Therefore, an EBNextract would be a viable substitute for individuals who do not produceendogenous EGF needed for human mesenchymal stem cells (hMSC) activationand differentiation to chondrocytes.

FIGS. 8 and 9 shows a comparison between an EBN extract product obtainedfrom the process described using Anti-N-glycans and/orAnti-Helix-Turn-Helix on human stem cell proliferation compared to anEBN extract from competitor A and B. The effect of the EBN extract toreplace EGF is much more significant for the EBN extract compared toboth competitor A and B products.

Effect of EBN's Extract on Influenza

Materials and Methods

1. Extraction Method

With reference to FIG. 1 , a piece of EBN weighing 10-50 g was washed inan enzymatic solution to remove mites for 5 minutes, the solution wasremoved. The bird's nest was washed with an aqueous solution containingnitrite reductase, the solution was removed. The cleaned EBN was driedfor 12 h at 70° C. The dried and cleaned EBN was grounded and siftedthrough a mesh (600 μm pore size) to remove plume and foreignsubstances. The grounded EBN was kept in distilled water (25 g/1000 mlwater) at 5° C. for 5 h and then heated at 121° C. for 10 min forsterilization.

The suspension was mixed with 270 ml of a solution containing anantibody for 20 min, and followed by 200 ml of acid at 4° C. overnight.The extract was homogenized with a homogenizer and heated at 100° C.After cooling to room temperature, the solution pH was adjusted to 7.0by dropwise addition of an alkali with stirring. The antibody boundedcompounds were released from the antibody by adding excess largerpeptides of natural glycoaminoglycans and cellular transcriptionalregulators. The released compounds are subsequently isolated from theadded peptides, enzymes and antibodies via the use of a dialysis bag.

The extract was treated with a corn or maize terminal protease at 45° C.for 1 hour at a pH of 6.5-9.0. The enzyme was inactivated by heating thesuspension to 70° C. for 5 min, and filtering off the precipitatedenzyme at a temperature above 55° C. The isolated products in thefiltrate was freeze dried for at least 24 hours.

For in vitro assays testing, the respective freeze dried EBN powder wasdissolved in distilled water to prepare a stock solution with aconcentration of 33.3 g/l and sterilized by gamma-radiation at 25 kGryfor 1 hour. Any insoluble matter was removed via centrifugation at 8000RPM. The stock solution was stored at −20° C. until required.

Soluble protein concentration in SF-EBN was determined by DC™ proteinassay (Bio-Rad, Cat. No. 5000116) and used bovine serum albumin as theprotein standard. Osmolarity and turbidity was measured by a vaporpressure osmometer (VAPRO) and microplate reader (Tecan Infinite M200)respectively.

In an alternative method, the bird's nests were washed for 10 min anddried for 12 h at 70° C. The dry bird's nests were ground and siftedthrough a mesh (600 μm pore size) to remove plume and foreignsubstances. The grounded bird's nests kept in distilled water (25 g/1000ml water) at 5° C. for 5 h and then heated at 121° C. for 10 min(sterilization). The suspension was mixed with 270 ml of the extractionsolution for 20 min and followed by 200 ml of acid at 4° C. overnight.The extract was homogenized with a homogenizer and heated at 100° C.

After cooling to the room temperature, the solution pH was adjusted to7.0 by dropwise addition of an alkali while stirring. The extract wastreated with the proprietary enzyme (the concentration depends on enzymespecifications) at 45° C. for 1 hour at pH 6.5-9.0. The enzyme was laterinactivated by heating the suspension to 70° C. for 5 min. The treatedextract was freeze dry. For in vitro assays testing, the freeze driedEBN powder was dissolved in distilled water at concentrations of 33.3g/l and sterilized by gamma-radiation at 25 kGry for 1 hour. Theinsoluble EBN extract was removed via centrifugation at 8000 RPM. Thesoluble fraction (SF-EBN) stored in −20° C. was used for influenzaneutralization studies.

Soluble protein concentration in SF-EBN was determined by DC™ proteinassay (Bio-Rad, Cat. No. 5000116) and used bovine serum albumin as theprotein standard. Osmolarity and turbidity was measured by a vaporpressure osmometer (VAPRO) and microplate reader (Tecan Infinite M200)respectively

2. Expansion and Creation of Vero Cell Bank

Vero cells (ATCC CCL-81) from a master cell bank at Passage 129 werethawed and expanded in serum-free media, OptiPro serum free medium (LifeTechnologies, Cat. No. 12309-019) supplemented with 4 mM L-glutamine(Life Technologies, Cat. No. 25030149) to generate a working cell bankwith 20 vials of 2×106 cells per vial. Vero cells were passaged every 4days using StemProAccutase (Life Technologies, Cat. No. A11105-01) forcell detachment and single cells generated were inoculated at celldensity of 1.5×104 cells/cm².

3. Effect of EBN Extract Solution on Vero Cell Growth

To test the effect of SF-EBN on cell growth, Vero cells were seeded at3×104 cells/well in 24-well tissue culture plate in OptiPro serum freemedium. One day post seeding, SF-EBN (33.3 g/l) was added to respectivewells to attain final concentrations of 3.3 g/l (10% v/v), 1.65 g/l (5%v/v), 0.83 g/l (2.5% v/v), and 0.33 g/l (1% v/v). Cell growth wasmonitored daily over 4 days. Cell counts were performed usingNucleocounter NC-3000 (Chemometec, Inc.) according to the recommendedprotocol.

4. Influenza Virus Propagation in Vero Cells

Vero cells were seeded at 2.5×104 cells/cm2 to obtain sub-confluenceafter 2 days of culture were infected with influenza A H1N1 virus(IVR-116, NIBSC code 06/108) and H3N2 (A/Wisconsin/67/2005 HGR, NIBSCcode 06/112)) at MOI (multiplicity of infection) of 0.001 to 0.01.Porcine trypsin (Sigma-Aldrich, Cat. No. T5266, 1500 BAEE unit/mg) usedfor the activation of influenza virus was prepared with deionized waterand filter sterilized to make a stock concentration of 5 mg/ml. Duringthe virus amplification, trypsin was added 30 minutes after inoculationwith the virus to obtain a final concentration of 5 μg/ml. Viruscontaining culture supernatant was harvested 2-3 days later (when 80%cytopathic effect (CPE) was observed) and centrifuged to remove celldebris. The virus stock solutions (H1N1 and H3N2) were stored in −80° C.Their virus titers were quantified using haemagglutination and tissueculture infectious dose (TCID50) assays as described below.

5. Quantification of Influenza Virus Titers

Virus titers were quantified using the haemagglutination assay andtissue culture infectious dose (TCID50) assay as described in Chen etal. (BMC Biotechnol., 2011, 11-81). For the haemagglutination assay, 4%human erythrocytes (Siemens Healthcare Diagnostics) were diluted inDulbecco's phosphate buffer solution (DPBS, Life Technologies, Cat. No.14190-250) to obtain a 0.75% cell suspension. The diluted erythrocytesuspension was then added to 2-fold serial dilutions of virus andcontrol samples. The highest dilution of virus which causes completehaemagglutination of erythrocytes is considered the HA titration endpoint. The HA titer (HAU/50 μl) is the reciprocal of the dilution ofvirus in the last well with complete haemagglutination (E.g. A dilutionof 2⁷ gives haemagglutination unit (HAU/50 μl) of 128).

TCID50 assay was performed in triplicates by adding 10-fold seriallydiluted virus samples to Vero cells cultivated in 96-well plates usingOptiPro serum free medium supplemented with 5 μg/ml of porcine trypsin.The plates were incubated for 3 days at 37° C. in a 5% CO2 atmosphereand then the cultures were checked under a light microscope forcytopathic effect. The dilution of the suspension that causes cytopathiceffects in 50% of the cultures (the median tissue culture infectivedose, TCID50/ml) was calculated according to the formula of Reed andMuench (1938).

6. Haemagglutination Inhibition Assay

Haemagglutination inhibition (HAI) assay was carried out using 96-wellmicrotiter plates as described previously (Guo et al., 2006, AntiviralRes., 70, 140-146). DPBS containing Mg2+ and Ca2+ was used as a dilutionbuffer. Human erythrocytes were used as indicator cells. Virussuspension (8 HAU/50 μl in 0.05 ml DPBS and 16 HAU/50 μl in 0.05 ml DPBSrespectively) was added to each well containing the EBN solution intwofold serial dilutions with the dilution buffer. 0.05 ml of 0.75%(v/v) human type 0 erythrocytes in DPBS was added to the plates andincubated for 1 h at 4° C. The maximum dilution of the samples showingcomplete inhibition of haemagglutination was defined as the HAI titer ofthe EBN solution.

7. Effect of EBN on Inhibition of Influenza Virus Replication

Vero cells were cultured in T25 flasks in OptiPro serum free medium withseeding density of 3×10⁴ cells/cm². When cell culture reached 90%confluency, the cultures were infected with influenza virus at MOI of0.001. SF-EBN was then added to reach final concentrations of 2 g/l(6.25% v/v), 0.26 g/l (0.78% v/v) and 0.03 g/l (0.10% v/v). After 2 h,SF-EBN containing culture medium was removed. The cells were washedthree times with OptiPro serum free medium. Porcine trypsin were addedto obtain a final concentration of 5 μg/ml. Culture supernatant werecollected after 24 h. SF-EBN antiinfluenza effect was validated viahaemagglutination and TCID50 assays.

8. Human Mesenchymal Stem Cells (hMSC) Cell Culture

hMSC (passage 8 to 9) were plated at a density of 2400 to 2800 cells/cm²in either T175 cm² cell culture flasks or Nunc™ EasyFill™ Cell Factory™Systems in MSC growth medium consisting of Minimum Essential Medium a,10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (all fromGibco). The medium was changed every 2 to 3 days. hMSC were passaged atabout 70% confluency when they were harvested using 0.25% Trypsin-EDTA(Gibco) for 5 mins at 37° C. Viability and cell count assays wereperformed with the automated NucleoCounter® NC-3000 (Chemometec). Allcultures were maintained at 37° C. in a 5% CO₂ humidified incubator(Thermo Scientific).

9. hMSC Cell Expansion for Cell Growth Curve

hMSC were plated at a density of 1000 cells/cm² in Nunclon™ DeltaSurface 24-well plates. hMSC was seeded at day 0 and cultured for 4days. hMSC grown only in BTI's proprietary serum-free MSC growth mediumwithout any growth factors and GeneOasis Pte Ltd's EBN extracts wereused as negative controls, and were known as “No Growth Factors”. hMSCgrown in BTI's proprietary serum-free MSC growth medium supplementedwith 10 ng/ml of PDGF (Peprotech), 5 ng/ml of TGFβ-1 (Peprotech), and 10ng/ml of FGFβ (Gibco) were also used as negative control, also referredto as “3 Growth Factors”. hMSC grown in BTI's proprietary serum-free MSCgrowth medium supplemented with 10 ng/ml of PDGF (Peprotech), 5 ng/ml ofTGFβ-1 (Peprotech), 1 ng/ml of EGF (Peprotech) and 10 ng/ml of FGFβ(Gibco) were used as positive control, also referred to as “4 GrowthFactors”. hMSC grown in serum-free MSC growth medium supplemented with 3growth factors (excluding EGF) with 10% EBN (vol/vol) were tested andwas referred to as “3 Growth Factors+10% EBN”. 10% (vol/vol) EBN wasadded on day 1. Cell count assays were performed daily with triplicatesfor each condition with the automated NucleoCounter® NC-3000(Chemometec). All cultures were maintained at 37° C. in a 5% CO2humidified incubator (Thermo Scientific).

10. Chondrogenic Differentiation

hMSC were grown as micromass pellets in clear round bottom ultra-lowattachment 96 well plates (Corning) for chondrogenic differentiation.Pellets were formed by centrifugation at 1000 rpm for 5 mins at roomtemperature (rt) using 2×10⁵ hMSC per pellet per well. They werecultured in chondrogenic differentiation medium containing DMEM-highglucose (Gibco), 1 mM sodium pyruvate (Gibco), 100 nM dexamethasone(Sigma), 0.1 mM L-ascorbic acid-2-phosphate (Sigma), 1% ITS+1 (Sigma),L-proline (Sigma) and 1% Penicillin/Streptomycin (Gibco). Pellets grownonly in chondrogenic differentiation medium without EBN extracts wereused as negative controls. Pellets grown in chondrogenic differentiationmedium with different concentrations of EBN (1.25%, 2.5%, 5% and 10%vol/vol) were tested. Pellets grown in chondrogenic differentiationmedium supplemented with 100 ng/ml of BMP2 were used as positivecontrols. Chondrogenic medium with or without supplementation waschanged every 2 to 3 days.

11. DNA, GAG and Collagen II Content Evaluation

Pellets (at least 3 per condition per timepoint) were rinsed once withPhosphate Buffer Saline (PBS) before immediate storage at −80° C. Afterthawing, the pellets were either digested with 0.125 mg/ml papain at 65°C. overnight for DNA and GAG quantification, or with 0.1 mg/ml pepsin at4° C. over 2 nights followed by 0.1 mg/ml elastase digestion at 4° C.overnight for Collagen II evaluation. DNA quantification was done usingQuant-iT Picogreen dsDNA Assay, GAG measurement was done using BlyscanSulfated Glycosaminoglycan Assay (Biocolor), and Collagen IIquantification was done by ELISA against Type II Collagen (Chondrex),all in accordance with manufacturer's instructions. All fluorometric andoptical readings were taken with a Tecan Infinite M200.

12. Statistical Analyses

Data were analyzed with statistical software Prism 6 (GraphPad).Multiple comparisons among different conditions were comparedstatistically using ordinary one-way analysis of variance (ANOVA) withTukey's multiple comparisons test. For all statistical tests, p valuesless than 0.05 were considered significant.

13. Comparison of GeneOasis EBN with Competitor A EBN and Competitor BEBN

hMSC were grown in a similar fashion as detailed in part 9. hMSC wasseeded at day 0 and cultured for 5 days. 2 types of comparison of theEBN extract to either Competitor A EBN or Competitor B EBN wereperformed. One type of comparison was to compare 10% of competitor EBNto GeneOasis EBN by normalizing or controlling for protein content (i.e.GeneOasis EBN control would have same amount of protein as that of therespective competitor). As measured, 10% of Competitor A EBN isequivalent to 4.39% of GeneOasis EBN. Thus, MSC growth mediumsupplemented with 3 growth factors (excluding EGF) with 4.39% GeneOasisEBN (vol/vol) were tested and was referred to as “GOA”. MSC growthmedium supplemented with 3 growth factors (excluding EGF) with 10% EBNof Competitor A (vol/vol) were tested and was referred to as “3 GrowthFactors+10% Competitor A”. Likewise for Competitor B, 10% of CompetitorB EBN is equivalent to 3.35% of GeneOasis EBN. Thus, MSC growth mediumsupplemented with 3 growth factors (excluding EGF) with 3.35% GeneOasisEBN (vol/vol) were tested and was referred to as “GOB”. MSC growthmedium supplemented with 3 growth factors (excluding EGF) with 10% EBNof Competitor B (vol/vol) were tested and was referred to as “3 GrowthFactors+10% Competitor B”. The second type of comparison was to compare10% of competitor EBN to GeneOasis EBN by normalizing or controlling forvolume (i.e. 10% GeneOasis EBN would be compared to 10% of CompetitorEBN by volume). The investigators are blinded to the identity ofCompetitor A and B throughout the execution and data analysis of therelated experiments.

In summary, we describe the novel EBN extraction method and found thatthe EBN extract of the present invention could inhibit thehaemagglutination of influenza A to human erythrocyte and reduceinfectivity in Vero cells. Using soluble fraction of EBN extract(SF-EBN), we showed that at the minimal concentrations of 0.5 g/l and 1g/L EBN could inhibit haemagglutination for H1N1 and H3N2 respectively.For the in vitro infectivity of influenza H1N1, we show that theaddition of SF-EBN at concentrations of between 0.03 to 2 g/l to theVero cell culture reduced the virus titer generated by at least 2 foldwhen compared to the culture without SF-EBN supplementation. CommercialEBN solutions were also tested in the haemagglutination inhibition assaywith influenza A. These EBN solutions were found to be either cytotoxicto the cells or did not exhibit any detectable anti influenza virusactivity. In conclusion, GeneOasis' proprietary EBN extract is potentand superior in the prevention of influenza virus infection.

Results

Prior to testing the effect of SF-EBN on cells and influenza virusreplication, we have carried out soluble protein assay with bovine serumas the standard as shown in Table 1 below. This will provide thereference point for future comparison between various sources andextraction methods of EBN.

TABLE 1 Properties of SF-EBN Properties Soluble protein content 2021μg/mL Osmolarity  348 mmol/kg Turbidity OD₆₀₀ 0.067 pH 8.6Effect of EBN on Vero Cell Growth

Four SF-EBN concentrations (3.3 g/l (10% v/v), 1.65 g/l (5% v/v), 0.83g/l (2.5% v/v), and 0.33 g/l (1% v/v)) were tested for their effect onVero cell growth in tissue culture plates. As shown in FIG. 2 , SF-EBNenhanced Vero cell growth in a concentration dependent manner whencompared to the control without SF-EBN. In the presence of 3.3 g/lSF-EBN, Vero cells reached the confluent cell density on day 3(^(˜)5×105 cells/cm2) earlier than the control (Day 4). The growthenhancement can be related to the presence of EGF-like molecules foundin EBN extract, where EGF is the known essential growth factor requiredby Vero cells for growth.

Inhibitory Effect of EBN Extract on Influenza Virus Infection

To determine the inhibitory effect of SF-EBN on the ability of thehaemagglutinin molecules located on the surface of the influenza virusto interact with erythrocytes, we have carried out haemagglutinationinhibition (HAI) assay, a commonly used assay for quantitating of theconcentration of influenza viruses. Two virus concentrations of 16 and 8HAU/50 μl were prepared, (8 HAU/50 μl is the recommended concentrationfound in WHO laboratory procedure for serological detection of influenzavirus). As shown in FIG. 3 , twofold serial dilutions of the SF-EBN(column 2-12) were mixed with human erythrocytes before the addition ofinfluenza viruses at two concentrations 16 and 8 HAU/50 μl (mentionedabove) in a 96-well plate. Erythrocytes that do not bind with influenzavirus would settle to the bottom of a well and form a red button. In thecase that the viruses bind to erythrocytes, a lattice was formed.Haemagglutination was not observed until 27 fold dilution for the twoinfluenza virus concentrations tested. The corresponding SF-EBNconcentration at that dilution is 0.5 g/l. This indicates that at thisconcentration of 0.5 g/l (but not lower) EBN blocks haemagglutination.The experiment was repeated using influenza H3N2 as shown in FIG. 4 .Higher SF-EBN concentrations of 2.1 g/L and 1.0 g/L were required toblock haemagglutination in the presence of H3N2 virus at concentrationsof 16 and 8 HAU/50 μl respectively. These results demonstrate that EBNextract can compete with the viral hemagglutinin molecules (measured byhaemagglutination of human erythrocytes) to reduce influenza virusinfectivity (similar to the activity of specific antiserum).

We also investigated the effect of SF-EBN on the inhibition of influenzavirus infection of Vero cells. Confluent Vero cells were infected withinfluenza virus H1N1 (M01 of 0.001) in the presence of SF-EBN of 2 g/l,0.26 g/l and 0.03 g/l. After 24 h post infection, the culturesupernatants were collected for virus titers quantification usinghaemagglutination assay. As shown in Table 2 below, SF-EBN concentrationof between 0.03 to 2 g/l showed about a twofold reduction in H1N1 virustiter. This indicates that EBN extract can reduce infectivity ofinfluenza virus (using Vero cells).

TABLE 2 Effect of SF-EBN on influenza virus H1N1 replication in Verocells. SF-EBN (g/l) Virus Titre (HAU/50 μl)^(#) Fold reduction 0 10.7 ±4.6  — 0.03 4 ± 0 2.6 0.26 5.3 ± 2.3 2   2 5.3 ± 2.3 2   ^(#)Result ofhemagglutination assays are derived from FIG. 5 Evaluation of SF-EBNfrom competitors for anti-viral effect

Two commercially available EBN solutions (Competitor A and B) wereselected for anti-viral activity determination. As shown in Table 3below, these commercial EBN solutions exhibited lower soluble proteinconcentration and osmolarity than SF-EBN.

TABLE 3 Physical properties of EBN solutions from competitor A and B.Soluble Protein Osmolarity Turbidity Concent (μg/ml) (mmol/kg) (OD₆₀₀)pH GeneOasis 2021 348 0.067 8.6 Competitor A  823 187 0.056 6.7Competitor B  628 220 0.056 7.3

Hence, SF-EBN was diluted with water to match the soluble proteinconcentration of the commercial EBN solutions for the haemagglutinationassay. Competitor A EBN solution was excluded from the assay as it wasfound to be cytotoxic to the cells, whereas the other EBN solutions(competitor B) showed no growth inhibition effect. The result of thehaemagglutination assay is shown in FIG. 6 . We found that thecommercial EBN solution (competitor B) did not exhibit any activity inpreventing H1N1 virus mediated haemagglutination of erythrocytes. Thisindicates this commercial EBN solution has no anti-viral properties asshown with SF-EBN. Since EBN is known to have anti-influenza virusactivity, we can conclude that the novel method for EBN extractiondeveloped by geniuses to be superior in the preservation of theantiviral property.

Effect of EBN's Extract on Human Neural Progenitor Cell Growth andDifferentiation

1. Material and Methods

1.1 Derivation and Maintenance of Human Neural Progenitors

Human neural progenitors (hNPC) were generated from human embryonic stemcell line (hESC) HES-3 by using the microcarrier suspension cultures.Subsequently, hNPCs were then grown on geltrex-coated plates, and fedwith NPC medium (Neurobasal Medium, 1×NEAA, 1× Penicillin-Streptomycin,2 mM L-Glutamine, 1× N2, 1× B27 minus Vitamin A, all from LifeTechnologies) supplemented with EGF (20 ng/ml, Peprotech) and FGF (20ng/ml, Peprotech). The medium was changed every other day. hNPCs werepassaged using StemPro Accutase (Life Technologies) when cells reachedabout 80-90% confluency.

1.2 Cell Growth and Cell Count of Human Neural Progenitors

hNPC were plated at a density of 2000 cells/well in geltrex-coated48-well plates, and grown for 1 week. hNPCs were fed with NPC mediumsupplemented with different combinations of growth factors (EGF, FGF)and GeneOasis Pte Ltd's EBN extracts. Cells were then lysed for totaland viable cell count using a nuclei count method with DAPI byNucleoCounter NC3000 (Chemometec) according to the manufacturer'sinstructions.

1.3 Cell Proliferation and Differentiation of Human Neural Progenitors

hNPC were plated at a density of 150,000 cells/well in geltrex-coated6-well plates, and grown for 1 and 3 weeks. hNPCs were fed with NPCmedium supplemented with different combinations of growth factors (EGF,FGF) and GeneOasis Pte Ltd's EBN extracts. During the culture period,hNPCs were passaged when cells reached 80% confluency. By the time point(1st week and 3rd week), hNPCs were dissociated into single cells withTryple (Life Technologies). Subsequently, cells were fixed,permeabilized, and incubated with primary antibodies, Anti-Ki-67 (1:200,BD Bioscience) for cell proliferation and anti-Tubulin R 3 (1:1000,Covance) for Neuron differentiation assessments. Alexa Fluor 488° goatantimouse (Life Technologies) was used as the secondary antibody. Allincubations were conducted at 4° C. for 20 min. Fluorescent measurementswere done using flow cytometer (GUAVA, Millipore). FlowJo software isused for the analysis of flow cytometry data.

2. Results

In this study, we investigated whether EBN extract can support theexpansion of hNPCs derived from human embryonic stem cells (hESCs). Asshown in FIG. 10 , hNPCs cultured in the presence of FGF-2 and EGFexhibited highest cell yield after 7 days. EBN extracts combined withgrowth factor FGF-2 achieved comparable cell growth profiles as thosewith FGF-2+EGF and FGF-2 alone conditions. Adding EBN without FGF-2 hasresulted in lower cell growth. The results suggested that the EBNextract cannot replace the FGF2, which is the most critical growthfactor for hNPC proliferation. On the other hand, even though EBNcontained EGF-like activities, which was shown in the report describingthe effect of EBN extract on proliferation of human mesenchymal stemcells in serum free medium, we observed no beneficial effect of EBNextract in the short term expansion of hNPCs in the presence of FGF-2.

We carried out further analysis of the expanded hNPCs. As shown in FIG.10B, the NPC culture with FGF-2 and EBN extract exhibited similarpopulation of cells expressing the cellular proliferation marker, ki67and neuronal differentiation marker, beta-Tubulin. EBN extractsupplemented culture without FGF-2 exhibited lower ki67 and highbeta-Tubilin cell population. The trend of ki67 was consisted to cellgrowth study where EBN extract could not support NPC growth withoutFGF-2. The low ki67 expression level is consistent to reported phenomenathat the decreased cell proliferation occurred at the onset of neuronaldifferentiation.

We have extended the study to three weeks. As shown in FIG. 11 , EBNextracts combined with FGF-2 seemed to exhibit higher cell yield thanthe cultures with FGF-2 only. The expressions of cell proliferationmarker ki67 were similar to the earlier culture in all conditions.However, we found that neuronal differentiation marker (beta-Tubulin)was significantly increased in hNPC culture supplemented with EBNextracts without FGF-2 and reached a similar level to the culturewithout both EGF and FGF-2. The increased Betatubulin positive cellpopulation indicated the development of neuronal cells. This wasconsistent with the cell morphology images shown in FIG. 12 , whereneuronal cell morphology (neurite extensions) became significant incultures with high beta-Tubilin expression. hNPC maintained theirtypical morphology when grown on the condition of 10% EBN extracts+ FGF.

In conclusion, our study indicates the present EBN extracts containEGF-like component, which can be beneficial to long term culturing ofhNPC. One of the future studies may be considered is the fractionationof the EBN extract to isolate the EGFlike component for furthercharacterization.

Effect of EBN's Extract on Cell Culture, Cell Growth and ChondrogenicAssays

1.2.1. hMSC Cell Culture

hMSC (passage 8 to 9) were plated at a density of 2400 to 2800 cells/cm²in either T175 cm² cell culture flasks or Nunc™ EasyFill™ Cell Factory™Systems in MSC growth medium consisting of Minimum Essential Medium a,10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (all fromGibco). The medium was changed every 2 to 3 days. hMSC were passaged atabout 70% confluency when they were harvested using 0.25% Trypsin-EDTA(Gibco) for 5 mins at 37° C. Viability and cell count assays wereperformed with the automated NucleoCounter® NC-3000 (Chemometec). Allcultures were maintained at 37° C. in a 5% CO2 humidified incubator(Thermo Scientific). See table below showing the well plate design.

Chondrogenic medium — +B2 +10% EBN +5% EBN +2.5% EBN +1.25% EBN 1 2 3 45 6 7 8 9 10 11 12 A D 7 D 21 D 7 D 21 D 7 D 21 D 7 D 21 D 7 D 21 D 7 D21 B D 7 D 21 D 7 D 21 D 7 D 21 D 7 D 21 D 7 D 21 D 7 D 21 C D 7 D 21 D7 D 21 D 7 D 21 D 7 D 21 D 7 D 21 D 7 D 21 D D 7 D 21 D 7 D 21 D 7 D 21D 7 D 21 D 7 D 21 D 7 D 21 E D 14 D 28 D 14 D 28 D 14 D 28 D 14 D 28 D14 D 28 D 14 D 28 F D 14 D 28 D 14 D 28 D 14 D 28 D 14 D 28 D 14 D 28 D14 D 28 G D 14 D 28 D 14 D 28 D 14 D 28 D 14 D 28 D 14 D 28 D 14 D 28 HD 14 D 28 D 14 D 28 D 14 D 28 D 14 D 28 D 14 D 28 D 14 D 281.2.2. hMSC Cell Expansion for Cell Growth Curve

hMSC were plated at a density of 1000 cells/cm² in Nunclon™ DeltaSurface 24-well plates. hMSC was seeded at day 0 and cultured for 4days. hMSC grown only in BTI's proprietary serum-free MSC growth mediumwithout any growth factors and GeneOasis Pte Ltd's EBN extracts wereused as negative controls, and were known as “No Growth Factors”. hMSCgrown in BTI's proprietary serum-free MSC growth medium supplementedwith 10 ng/ml of PDGF (Peprotech), 5 ng/ml of TGFß-1 (Peprotech), and 10ng/ml of FGFß (Gibco) were also used as negative control, also referredto as “3 Growth Factors”. hMSC grown in BTI's proprietary serum-free MSCgrowth medium supplemented with 10 ng/ml of PDGF (Peprotech), 5 ng/ml ofTGFR-1 (Peprotech), 1 ng/ml of EGF (Peprotech) and 10 ng/ml of FGFß(Gibco) were used as positive control, also referred to as “4 GrowthFactors”. hMSC grown in serum-free MSC growth medium supplemented with 3growth factors (excluding EGF) with 10% EBN (vol/vol) were tested andwas referred to as “3 Growth Factors+10% EBN”. 10% (vol/vol) EBN wasadded on day 1. Cell count assays were performed daily with triplicatesfor each condition with the automated NucleoCounter® NC-3000(Chemometec). All cultures were maintained at 37° C. in a 5% CO2humidified incubator (Thermo Scientific).

1.2.3. Chondrogenic Differentiation

hMSC were grown as micromass pellets in clear round bottom ultra-lowattachment 96 well plates (Corning) for chondrogenic differentiation.Pellets were formed by centrifugation at 1000 rpm for 5 mins at roomtemperature (rtm) using 2×105 hMSC per pellet per well. They werecultured in chondrogenic differentiation medium containing DMEM-highglucose (Gibco), 1 mM sodium pyruvate (Gibco), 100 nM dexamethasone(Sigma), 0.1 mM L-ascorbic acid-2-phosphate (Sigma), 1% ITS+1 (Sigma),L-proline (Sigma) and 1% Penicillin/Streptomycin (Gibco). Pellets grownonly in chondrogenic differentiation medium without the presentinvention's extracts were used as negative controls. Pellets grown inchondrogenic differentiation medium with different concentrations of EBN(1.25%, 2.5%, 5% and 10% vol/vol) were tested. Pellets grown inchondrogenic differentiation medium supplemented with 100 ng/ml of BMP2were used as positive controls. Chondrogenic medium with or withoutsupplementation was changed every 2 to 3 days.

1.2.4. DNA, GAG and Collagen II Content Evaluation

Pellets (at least 3 per condition per timepoint) were rinsed once withPhosphate Buffer Saline (PBS) before immediate storage at −80° C. Afterthawing, the pellets were either digested with 0.125 mg/ml papain at 65°C. overnight for DNA and GAG quantification, or with 0.1 mg/ml pepsin at4° C. over 2 nights followed by 0.1 mg/ml elastase digestion at 4° C.overnight for Collagen II evaluation. DNA quantification was done usingQuant-iT Picogreen dsDNA Assay, GAG measurement was done using BlyscanSulfated Glycosaminoglycan Assay (Biocolor), and Collagen IIquantification was done by ELISA against Type II Collagen (Chondrex),all in accordance with manufacturer's instructions. All fluorometric andoptical readings were taken at with Tecan Infinite M200.

1.2.5. Statistical Analyses

Data were analyzed with statistical software Prism 6 (GraphPad).Multiple comparisons among different conditions were comparedstatistically using ordinary one-way analysis of variance (ANOVA) withTukey's multiple comparisons test. For all statistical tests, p valuesless than 0.05 were considered significant.

1.2.6. Comparison of the Present EBN Extract with Competitor A EBN andCompetitor B EBN

hMSC were grown in a similar fashion as detailed in 1.2.2. hMSC wasseeded at day 0 and cultured for 5 days. 2 types of comparison of thepresent EBN extract to either Competitor A EBN or Competitor B EBN wereperformed. One type of comparison was to compare 10% of competitor EBNto the present EBN extract by normalizing or controlling for proteincontent (i.e. the present EBN extract control would have same amount ofprotein as that of the respective competitor). As measured, 10% ofCompetitor A EBN is equivalent to 4.39% of the present EBN extract.Thus, MSC growth medium supplemented with 3 growth factors (excludingEGF) with 4.39% GeneOasis EBN (vol/vol) were tested and was referred toas “GOA”. MSC growth medium supplemented with 3 growth factors(excluding EGF) with 10% EBN of Competitor A (vol/vol) were tested andwas referred to as “3 Growth Factors+10% Competitor A”.

Likewise for Competitor B, 10% of Competitor B EBN is equivalent to3.35% of the present EBN extract. Thus, MSC growth medium supplementedwith 3 growth factors (excluding EGF) with 3.35% of the present EBNextract (vol/vol) were tested and was referred to as “GOB”. MSC growthmedium supplemented with 3 growth factors (excluding EGF) with 10% EBNof Competitor B (vol/vol) were tested and was referred to as “3 GrowthFactors+10% Competitor B”. The second type of comparison was to compare10% of competitor EBN to the present EBN extract by normalizing orcontrolling for volume (i.e. 10% of the present EBN extract would becompared to 10% of Competitor EBN by volume). The investigators areblinded to the identity of Competitor A and B throughout the executionand data analysis of the related experiments.

The table below is a summary of methods of comparison of the present EBNextract to either Competitor A EBN or Competitor B.

GeneOasis vs Competitors A&B Aim-To investigate if 10% of competitor Aand B have the same effect of Gene Oasis EBN on cell growth in Serumfree conditions (Normalized to total protein in media (ug/ml) or volumeratio of EBN in media) Conditions Cell line: hMSC Media: In-house serumfree media Controls 1) 4 growth factors (Positive control) 2) No growthfactor (Negative control) 3) 3 growth factor only-No EGF (Baselinecontrol) 3) Gene oasis control for compethor A (GOA)-Same amount ofprotein as Competitor A 4) Gene oasis control for competitor B(GOB)-Same amount of protein as Competitor B According to ProteinConcentrations from Gerine 10% Competitor A EBN equivalent to 4.39% GeneOasis EBN (GOA) 10% Competitor B EBN equivalent to 3.35% Gene Oasis EBN(GOB)2. Results

The present EBN extract can elicit an increase in human stem cellproliferation during cell expansion in vitro, as shown in the FIGS. 13to 24 .

Whilst there has been described in the foregoing description preferredembodiments of the present invention, it will be understood by thoseskilled in the technology concerned that many variations ormodifications in details of design or construction may be made withoutdeparting from the present invention.

The invention claimed is:
 1. A bird's nest extract comprising at leastone molecule obtained from edible bird's nest (EBN) obtained from amethod comprising the steps of: (a) washing raw EBN; (b) filtering thewashed EBN; (c) extracting the molecule from the EBN, wherein extractingthe molecule is carried by exposing the washed EBN to any one of anextraction solution selected from the group consisting of: (i) asolution comprising an anti-N glycan and an anti-O glycan; (ii) asolution comprising an anti-heparan sulphate, an anti-chondroitin, ananti-keratan, and an anti-dermatan; and (iii) a solution comprising ananti-leucine zipper, an anti-helix-turn-helix, and an anti-zinc finger.2. A composition comprising an extract according to claim
 1. 3. Thecomposition according to claim 2 further comprising a maltodextrin. 4.The composition according to claim 3, wherein the amount of maltodextrinpresent in the composition is between 30 wt % to 75 wt %.
 5. Thecomposition according to claim 1 for use in medicine.
 6. Apharmaceutical composition comprising an extract according to claim 1and a pharmaceutically-acceptable carrier, excipient or diluent.